PET, being a molecular imaging technology, detects a myriad of diseases non-invasively. PET imaging systems create images based on the distribution of positron-emitting isotopes in the tissue of a patient. The isotopes are typically administered to a patient by injection of probe molecules that comprise a positron-emitting isotope, such as F-18, C-11, N-13, or O-15, covalently attached to a molecule that metabolizes or localizes in the body (e.g., glucose) or that binds to receptor sites within the body. In some cases, the isotope is administered to the patient as an ionic solution or by inhalation. Clinicians employ PET to accurately detect, stage, and restage cancer in patients. One of the most widely used positron-emitter labeled PET molecular imaging probes is 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Because early changes in glucose utilization have been shown to correlate with outcome predictions, clinicians use PET-FDC imaging to monitor cancer chemo- and radiotherapy. Other molecularly targeted PET imaging tracers are being developed to image other enzyme-mediated transformations in cancer tissue. Ongoing research efforts are directed at identifying additional biomarkers that show a very high affinity to and specificity for, tumor targets to support cancer drug development and to provide health care providers with a means to accurately diagnose disease and monitor treatment. Such imaging probes can dramatically improve the apparent spatial resolution of the PET scanner, allowing smaller tumors to be detected, and nanomole quantities to be injected in patients.
While the clinical use of PET for detecting cancer is growing, FDG based imaging does have limitations. Accumulation in inflammatory tissue limits the specificity of FDG-PET. Conversely, nonspecific FDG uptake may also limit the sensitivity of PET for tumor response prediction. Therapy induced cellular stress reactions have been shown to cause a temporary increase in EDG-uptake in tumor cell lines treated by radiotherapy and chemotherapeutic drugs. Further, physiological high normal background activity (i.e., in the brain) can render the quantification of cancer-related FDG-uptake impossible in some areas of the body. Ongoing research efforts are directed to identifying additional biomarkers that show a very high affinity to, and specificity for, tumor targets to support cancer drug development and to provide health care providers with a means to accurately diagnose disease and monitor treatment. Such imaging probes can dramatically improve the apparent spatial resolution of the PET scanner, allowing smaller tumors to be detected, and nanomole quantities to be injected in patients. A promising area for the development of these new agents focuses on imaging the enzymes associated with cellular proliferation. More specifically, new PET imaging agents that mimic the natural nucleoside thymidine offer the opportunity to directly observe the enzymatic pathways associated with thymidine metabolism and cell proliferation.
The nucleoside thymidine (FIG. 1) fulfills a vital role in cell growth via its participation in DNA replication and cell division. The cellular recruitment and utilization of thymidine occurs via discreet pathways. Nucleoside transporters ENT1 and/or CNT1 shuttle thymidine from the extracellular matrix into cells. In certain cancer cells, nucleoside transporter expression increases several fold to meet the demand for DNA synthesis and thymidine utilization. Once inside the cell, human thymidine kinase-1 (hTK-1) metabolizes thymidine into its 5′-O-monophosphate derivative, preparing it for incorporation into the growing DNA chain. The rate of thymidine metabolism varies depending on the stage of cell growth. In the resting phase, the consumption and incorporation of thymidine by the cell slows as DNA replication also slows. However, in the waking phase, or S phase, over-expression of hTK-1 is needed to metabolize the larger quantity of thymidine transported across the cell membrane.
Proliferating cancer cells increase their demand for thymidine by up-regulating both nucleoside transporters and hTK-1. The difference in thymidine uptake between cancer cells and normal cells translates into preferential imaging of the metabolic fate of thymine analogs in cancer cells in the presence of normal cells. Thus, radiotracers undergo transport across the cellular membrane, although the specific mechanism of action of this transport remains unknown, followed by 5′-O-phosphorylation metabolism by thymidine kinase. The metabolizing phosphorylation step effectively traps the tracer intracellularly and preferential accumulation of the tracer builds within cancer cells. This targeted accumulation leads to visualization of cancer cells in the presence of normal cells.
The fluorinated analog of thymidine 3′-[8F]Fluoro-3′-deoxythymidine (FLT), FIG. 2, successfully visualizes thymidine kinase activity in vivo. Non-radioactive FLT was originally developed as an antiviral then as a potential therapeutic for HIV; however, its toxicity profile prevented its therapeutic use. Fortunately, radiolabeled FLT showed promise as an imaging agent for detecting cancer. Radiolabeled FLT uptake by cancer cells correlates well with tumor proliferation and can be used to monitor response to therapy. Clinicians use radiolabeled FLT to assist in detecting and staging lung tumors and brain gliomas. More specifically, FLT imaging can characterize malignant from benign tumors and determine proliferation rates of lung tumors. For use in detecting and staging non-small cell lung cancers, FLT may provide additional information regarding the risk of recurrence after resection.
FLT successfully mimics thymidine recruitment and metabolism in vivo, although there are exceptions. For instance, while hTK-1 recognizes FLT as a substrate, mitochondrial thymidine kinase-2 (TK-2) does not and metabolizes FLT poorly. This serendipitous selectivity aids the detection of hTK-1 activity in vivo even in the presence of low levels of mitochondrial TK-2 expression. FLT also displays a favorable metabolic profile relative to another DNA proliferation marker, 11C-thymidine. Unlike the rapid catabolism of phosphorylated 11C-thymidine into various metabolites, FLT-phosphate resists thymidine phosphorylase catabolism. This metabolic stability simplifies the interpretation of PET images as there are fewer metabolites present. 11C-thymidine based PET imaging requires complicated mathematical models for image interpretation to compensate for 11C-thymidine derived metabolites. FLT exhibits a fairly typical biodistribution pattern: it localizes in the bladder and kidneys, which excrete undegraded FLT. However, the liver retains FLT although mainly as the glucoronidated species. FLT also distributes amongst haematopoietic bone marrow, a site of high cellular proliferation. On average, the injected dose per gram of FLT in tumors is 5%.
Despite the clinical success of FLT, imaging with FLT does have certain drawbacks. First, and most importantly, FLT uptake in tumors consistently remains lower than FDG uptake. This low tumor uptake has made imaging difficult because of the low signal to noise ratios. Additionally, the high hepatic retention of FLT prevents accurate imaging of tumors and metastases near the liver thus limiting the utility of FLT in the clinical setting.
FMAU (FIG. 3) is another nucleoside analog that has shown promise as a proliferative marker. While its uptake in cancer cells appears promising, the multistep radiosynthetic method for preparing this molecule severely limits its use as a standard clinical biomarker.
Previous work by Eriksson and coworkers offered the initial direction towards designing new thymidine-based imaging agents. They discovered that 3-N boronated thymidine analogs containing large closo-carboranylalkyl groups served as boron neutron capture therapy agents (FIG. 4). In addition to using the boron cage as a sink for incoming neutrons, several pharmacokinetic benefits happen to make these thymidine analogs more successful than anticipated. First, the boron group adds sufficient lipophilicity thus aiding in its transport across cell membranes. Secondly, because of the steric bulk at the 3-N position, these boronated compounds remained substrates for hTK-1 but not hTK-2. These results confirm the finding that hTK-1 tolerates bulky substituents preferentially over TK-2. These compounds are also taken up and retained by hTK-1 expressing cells in vitro but not retained in non-TK-1 expressing cells. While these carboranylated thymidines cannot be used directly for PET imaging, they do provide insight into other thymidine analogs that may image hTK-1 activity in vivo.